大二课件1Chapter4Proteinproperties,classificationandresearchmethods副本.pptVIP

Chapter 4 Protein Properties, Classification Research Methods Outline Physical and Chemical Properties of Proteins Research methods of proteins Classification of proteins (self-study) Solid-phase synthesis of polypeptides (self-study) Loss of Biological Activity Loss of Solubility Increased Proteolysis More sensitive to protease Increased viscosity  Acid-catalyzed hydrolysis: can destroy Trp; Gln→Glu, Asn→Asp Base-catalyzed hydrolysis: can cause racemation of amino acids Enzymatic hydrolysis * UV absorption: the absorbance peak at 280nm pI value and solubility Denature and Re-nature Hydrolysis Other properties Some Properties of Proteins Disruption of the normal 3D structure of a protein, such that it loses biological activity. Usually caused by heat, Chemicals or changes in pH. Usually irreversible. A cooked egg cannot be “uncooked.” Denaturation Measuring Protein Denaturation Protein Hydrolysis Lets say you discover a new protein: protein X You might ask the following questions: How big is the protein? What is its isoeletric point? Is it present in other cells / organisms? What is its sequence? What is its structure? Experimenting with proteins… Method: electrophoresis Separates protein molecules based on their sizes 1. How big is protein X? Procedure: Incubate protein X with SDS so that it becomes negatively charged Load onto gel Apply voltage to the gel Stain gel with a protein dye (e.g. Coomasie Blue) This particular kind of electrophoresis is also called SDS(Polyacrylamide Gel Electrophoresis) Electrophoresis Molecular Weight Markers Protein X Electrophoresis 2. What is protein Xs isoelectric point? Method: isoelectric focusing Separates protein molecules based on their isoelectric point Remember proteins have different charges at different pH? Procedure: Make a gel with pH gradient Load protein Find the pH where the protein stops moving High Method: Western blotting Cells contain many diff